This project is focused on the role of local factors in the accelerated bone turnover following estrogen withdrawal leading to bone loss and osteoporosis. IL-1 and TNF have been implicated in this process by studies showing that bone loss in ovariectomized mice can be blocked by IL-1 and TNF inhibitors. It is hypothesized that increased IL-1 TNF activity after estrogen withdrawal may inhibit osteoblastogenesis and reduce the formation response to resorption. Using a murine model of estrogen withdrawal, we will examine production of both spontaneous and induced IL-1 and TNF in bone and bone marrow, as well as IL-1 and TNF bioactivity in marrow supermatants. Mice will be sham-operated, with placement of placebo pellets, and ovariectomized, with placement of placebo or estradiol pellets, and sacrificed at varying times thereafter, mRNAs for IL-1alpha and beta, IL-1ra -, IL-1 receptor types l and ll, TNF, and TNF receptor types l and ll will be analyzed. Cytokine levels will be determined by ELISA in bone marrow supernatants. Mice transgenic for IL-1 or TNF promoter-CAT reporter constructs will also be studied. To assess effects of estrogen withdrawal on osteoprogenitors, marrow stromal cells (MSC) from these mice will be cultured for 12 and 21 d and analyzed for alkaline phosphatase positive colonies and mineralizing nodules. To asses the roles of IL-1 and TNF, we will examine their in vitro effects on cultured MSC and the effects of IL-1 and TNF inhibitors on exvivo MSC cultures from estrogen withdrawn mice. MSC from mice deficient for IL-1 and TNF receptors will also be studies. Effects on MSC differentiation of marrow supernantants from SHAM, OVX and OVX plus E mice will be examined plus/minus IL-1 and TNF inhibitors. Finally, we will develop constructs to target overexpression of IL-1ra and TNFbp to bone using the 3.6 COL1A1 promoter. After in vitro testing, transgenic mice lines will be developed for each inhibitor and bred together to develop lines expressing both inhibitors. Bone loss in overexpressing mice and wild type controls will be compared after estrogen withdrawal by static and dynamic histomorphometry.